We study the mechanism and regulation of protein synthesis in eukaryotic cells focusing on regulation by GTP-binding proteins and protein phosphorylation. The first step of protein synthesis is binding the initiator Met-tRNA to the small ribosomal subunit by the factor eIF2. The eIF2 is a GTP-binding protein and during the course of translation initiation the GTP is hydrolyzed to GDP. The eIF2 is released from the ribosome in complex with GDP and requires the guanine-nucleotide exchange factor eIF2B to convert eIF2-GDP to eIF2-GTP. This exchange reaction is regulated by a family of stress-responsive protein kinases that specifically phosphorylate the alpha subunit of eIF2 on serine at residue 51, and thereby covert eIF2 into an inhibitor of eIF2B. Among the family of eIF2alpha kinases are GCN2, which is activated under conditions of amino acid starvation, PKR, which is activated by double-stranded RNA and downregulates protein synthesis in virally infected cells, and PERK, activated under conditions of ER stress. In collaboration with Frank Sicheri we determined the structure of the PKR kinase domain in complex with eIF2alpha. This structural analysis revealed that eIF2alpha binds to the C-terminal lobe making intimate contact with helix alphaG, while catalytic domain dimerization is mediated by a back-to-back orientation of the kinase N-terminal lobes (reference 3). Positioning of the eIF2alpha aspartate-83 residue near PKR helix alphaG places the serine-51 residue near the active site of the kinase. Consistent with the structural data, mutations in PKR helix alphaG specifically impair phosphorylation of eIF2alpha. Moreover, mutations that activate PKR map to the catalytic domain dimer interface and promote kinase domain dimerization. Conversely, charge-reversal mutations that disrupt a conserved salt-bridge in the dimer interface block PKR autophosphorylation and eIF2alpha phosphorylation. Importantly, combining the two charge-reversal mutations in the same PKR allele, designed to restore the salt-bridge interaction with opposite polarity, rescued PKR activity. Finally, mutation of the conserved threonine-446 autophosphorylation site in PKR impairs eIF2alpha phosphorylation and viral pseudosubstrate binding. We propose an ordered mechanism of PKR activation in which catalytic domain dimerization triggers autophosphorylation and specific substrate recognition (reference 4). Interestingly, the residues forming the salt-bridge interaction in the PKR dimer interface are conserved among the eIF2alpha kinases. The corresponding single mutations designed to disrupt the putative salt-bridge interactions in GCN2 and PERK abolished kinase activity. More importantly, the double mutations in GCN2 and PERK, which would restore the putative salt-bridge interactions, restored the kinases' function both in vivo and in vitro. We conclude that the back-to-back dimer orientation observed in the PKR crystal structure is critical for the activity of PKR, GCN2 and PERK and that PKR structure represents the active state of the eIF2alpha kinase domain.[unreadable] The translation initiation factor eIF2 is composed of three polypeptide chains that assemble to form a stable complex. The gamma subunit of eIF2 contains a consensus GTP-binding (G) domain, and the factor must bind GTP to form a stable eIF2?GTP?Met-tRNA ternary complex. The GTPase-activating protein (GAP) eIF5 promotes GTP hydrolysis by eIF2 and the guanine-nucleotide exchange factor (GEF) eIF2B is responsible for exchanging GTP for GDP on eIF2 enabling the factor to function in additional rounds of translation initiation. GST pull-down experiments revealed that eIF2alpha, eIF2beta, eIF5 and eIF2B interacted with full-length eIF2gamma, whereas eIF5 and eIF2B, but not eIF2alpha or eIF2beta, bound to the eIF2gamma G domain. Importantly, these interactions were mapped to the catalytically critical N-terminus of eIF5 and C-terminal domain of eIF2Bepsilon. Thus, these critical regulators of eIF2 function make direct contacts with the G domain of eIF2gamma, consistent with their roles to promote GTP hydrolysis and GTP-GDP exchange on eIF2 (reference 2).[unreadable] The GTP-binding protein eIF5B catalyzes ribosomal subunit joining in the final step of translation initiation. The eIF5B is an ortholog of prokaryotic translation initiation factor IF2. Previous studies revealed that eIF5B consists of four domains that structurally assemble to form a chalice-shaped molecule. The G domain plus domains II and III form the cup of the chalice, a long alpha helix forms the stem, and domain IV is the base of the chalice. In addition, we previously showed that the domain IV of eIF5B binds to the C-terminal tail of the factor eIF1A (an ortholog of prokaryotic factor IF1). The eIF5B-eIF1A interaction is critical for efficient ribosomal subunit joining (reference 1). We propose that the eIF5B-eIF1A interaction promotes eIF5B recruitment to the ribosome and also facilitates release of the factors following GTP hydrolysis by eIF5B. Mutation of the conserved threonine residue in the switch 1 element of the eIF5B GTP-binding domain abolished GTP hydrolysis, but did not impair subunit joining in vitro. Intragenic suppressors of the switch 1 mutation uncoupled eIF5B GTPase and translational stimulatory activities indicating a regulatory rather than mechanical role for eIF5B GTP hydrolysis in translation initiation. We propose that in the presence of GTP eIF5B binds the ribosome and promotes subunit joining, which in turn triggers GTP hydrolysis leading to the factor's release from the ribosome. Mutation of the conserved glycine in switch 2 of eIF5B impaired GTP binding, GTP hydrolysis, translation initiation and yeast cell growth. Intragenic suppressors of the slow-growth phenotype associated with the switch 2 mutation mapped to switch 1 and to helix 8 (linking domains II and III). The intragenic suppressors restored both the GTP binding and GTPase activities of eIF5B revealing that the universally conserved glycine in switch 2 is not absolutely essential. Interestingly, the intragenic suppressors in switch 1 and helix 8 are located close to contact sites with switch 2, and the suppressor mutations are predicted to allosterically affect the position of switch 2. We propose that mutation of the conserved glycine in switch 2 alters the structure of the eIF5B active site, and that the two intragenic suppressor mutations restore a favorable geometry to the eIF5B active site by re-positioning switch 2 into a preferred location. As the switch 2 mutation and the switch 1 suppressor mutation map to elements conserved in all GTP-binding proteins, we believe that this interaction may be of importance for all GTP-binding proteins.